D1 Protein Degradation and psbA Transcript Levels in Synechocystis PCC 6803 during Photoinhibition in vivo

J. Plant Physiol.

Vol. 142. pp. 669-675 (1993)

D1 Protein Degradation and psbA Transcript Levels in Synechocystis pee 6803 during Photoinhibition in vivo ElRA KANERVO, PIRKKO MAENPAA,

and

EVA-MARl ARO

Department of Biology, University of Turku, SF-20500 Turku, Finland Received April 15, 1993 . Accepted June 19, 1993

Summary

The relation of photo inhibition of Photosystem II (PS II) to the rate of degradation of the reaction center protein D1 was studied in vivo in the cyanobacterium Synechocystis 6803. Exposure of cells to a PPFD of 1500 Ilmol m -2 s -1 induced 75 % photoinhibition of oxygen evolution of PS II within 2 h. In spite of severe photoinhibition, only a slight net decrease was observed at the steady-state level of the D1 protein as determined by quantitative immunoblotting analysis. However, in the presence of protein synthesis inhibitors degradation of the D1 protein occurred rapidly and photo inhibition of PS II was accelerated. Pulse and chase experiments under strong illumination revealed that the D1 protein turned over rapidly with a half-life of about 30 min. The presence or absence of psbA mRNA carrying polyribosomes on the thylakoid membranes did not significantly affect the rate of D1 protein degradation. Our results indicate that no direct synchronization exists between degradation and synthesis of the D1 protein and it is probable that reassembly and activation of PS II after insertion of a new copy of the D1 protein to the PS II complex are the rate-limiting factors of the repair of photodamaged PS II in cyanobacteria. Northern blot analysis of psbA mRNA in the presence and absence of protein synthesis inhibitors demonstrated that protein factors are needed to regulate the expression of the psbA genes in Synechocystis 6803.

Key words: Synechocystis 6803, cyanobacterium, Dl-protein, photoinhibition, Photosystem II, psbA-gene. Abbreviations: CAP = D-threo-chloramphenicol; DCBQ = 2,6-dichloro-p-benzoquinone; kb = Kilobase; kDa = Kilodalton; PAGE = Polyacrylamide gel electrophoresis; PPFD '" Photosynthetic photon flux density; PS II = Photosystem II; PVDF '" Polyvinylidene difluoride; SDS = Sodium dodecyl sulphate. Introduction

Exposure of oxygenic photosynthetic organisms to high light causes photoinhibition of photosynthesis (Powles, 1984). Photoinhibition is associated with an inactivation of PS II electron transport and subsequent degradation of the D1 reaction center protein of PS II (Virgin et al., 1988; Aro et al., 1993). The D1 protein, encoded by the chloroplast psbA gene, has been shown to exhibit a high rate of light dependent turnover in vivo (Mattoo et al., 1984), which is enhanced by photo inhibitory light intensities (Ohad et al., 1984; Schuster et al., 1988; Prasil et al., 1992). The light-dependent degradation of the D1 protein is thought to be due to a membrane-bound endoproteolytic activity of PS II (Vir© 1993 by Gustav Fischer Verlag, Stuttgart

gin et al., 1988), but the exact mechanism has not been elucidated yet. The expression of the psbA gene is regulated by light at all levels of protein synthesis (Erickson and Rochaix, 1992). In cyanobacteria, the light control of psbA gene expression operates mainly at the level of transcription (Mohamed and Jansson, 1989; Schaefer and Golden 1989), whereas translational regulation seems to be the crucial control level in algae and plants (Fromm et al., 1985; Klein, 1991). Photosynthetic organisms can survive photo inhibition through an efficient repair system of PS II. During the repair cycle, partial disassembly and reassembly of PS II complex occurs: in plants and the green algae, reaction center core complexes move from the appressed thylakoids to the non-

670

ElRA KANERVO, PIRKKO MAENPAA, and EVA-MARl ARO

appressed thylakoid region, where degradation and replacement of the D1 protein occur (Adir et aI., 1990; Ghirardi et aI., 1990; Melis, 1991). Synthesis of the D1 protein takes place in polyribosomes attached to the non-appressed thylakoids (Herrin and Michaels, 1985; Mattoo and Edelman, 1987). Re-establishment of functional PS II requires C-terminal processing and acylation of the newly synthesized D1 protein as well as reattachment of released polypeptides and cofactors of PS II (Mattoo and Edelman, 1987; Virgin et aI., 1988; Aro et aI., 1993). According to our recent hypothesis, thylakoid stacking is supposed to regulate the D1 protein turnover by preventing the premature degradation of the D1 protein in stacked areas, where resynthesis is not possible (Kettunen et al., 1991; Tyystjarvi et aI., 1992). In cyanobacteria, thylakoid membranes are not organized into appressed and non-appressed regions as in chloroplasts (Andersson and Anderson, 1980; Stanier (Cohen-Bazire), 1988). This fact made us assume that cyanobacterial thylakoid organization probably allows an efficient degradation and resynthesis of the D1 protein. In the present work, our aim was to study the in vivo relationship between the rate of degradation of the D1 protein and the rate of photoinhibition of PS II in cyanobacterial cells. We also wanted to clarify whether there is any synchronization between degradation and synthesis of the D1 protein and whether thylakoid-bound polyribosomes play any role in this respect.

Measurement of oxygen evolution ofPS II Electron transport activity of PS II was measured from intact cells at 32°C using a Clark-type oxygen electrode in saturating (3000~molm-2s-l) light intensity. 0.25mM DCBQ was used as an electron acceptor with 0.25 mM ferricyanide (Tae and Cramer, 1992). Chlorophyll concentration from intact cells was determined according to Bennett and Bogorad (1973).

In vivo pulse and chase experiments and protein analysis

p5S]L-methionine (1000Ci/mmol, Amersham) was added to the culture (1 0 ~g chi mL -I) to a concentration of 1 ~M. Radiolabeling was carried out for 1 h at the PPFD of 1500 ~molm-2s-1 in the conditions described for photoinhibition (see above) in the absence of antibiotics. Subsequently, the cells were transferred to new BG-ll medium containing 1 mM non-radioactive L-methionine and the high-light incubation was continued either in the absence or presence of antibiotics. The chase samples were taken at the time points 0, 20, 40, 60 and 120 min. Thylakoid membrane isolation was performed at 4°C in dim light. The cells were broken by vortexing with glass beads in STN buffer (10mM Tris-HCI, pH 8.0, OAM sucrose, 10mM NaCI and 20 mM NaEDTA) and the homogenate was centrifuged (700 x g, for 5 min). Thylakoid membranes from the resulting supernatant were pelleted (15,500 x g, for 15 min) and resuspended in STN buffer. The chlorophyll concentration from the thylakoid suspension was determined in 80 % acetone according to Arnon (1949). Thylakoid polypeptides were solubilized and separated by denaturing SDS-PAGE, 12-22.5% (w/v), according to Laemmli (1970). The samples were loaded on chlorophyll basis. The gels were fixed, treated with amplifier (AmplifyTM, Amersham) and dried. Autoradiographic signals were measured by a laser densitometer (LKB) and the samples were normalized using the sum of several stable polypeptide bands as a reference.

Materials and Methods

Culture conditions The cyanobacterium Synechocystis 6803 was grown in BG-ll medium (Williams, 1988) at 32°C under constant illumination at a PPFD of 40~molm-2s-1 (Philips TLD 36W/86 tubes). The cells were grown gently shaken and the cultures used for experiments were in the logarithmic growth phase.

Conditions for photoinhibition The cells were transferred to fresh BG-ll medium at a concentration of 10 ~gchl mL -I. Cell suspension was illuminated at a PPFD of 1500~molm-2s-1 at 32°C with gentle stirring. In the presence of rifampicin, the PPFD was adjusted to compensate for the integrated absorption of rifampicin through the whole suspension. This adjustment required a 85 % higher PPFD value at the surface of a rifampicin-containing sample, when the optical path length of the suspension was 10 mm. A slide projector lamp was used as a light source. Antibiotics were added to the cell suspensions at the same time as the photoinhibition treatment started, if not otherwise mentioned (see Figure legends). D-threo-chloramphenicol (CAP, Sigma), streptomycin (Boehringer-Mann~eim) and rifampicin (Sigma) were used at concentrations of 200, 250 and 500 ~g mL -I, respectively. Since rifampicin was subjected to photodegradation, an addition of 200 ~g mL -1 was made once every 25 minutes. We also checked that photodegradation products of rifampicin possibly formed did not change the transmission spectrum of this antibiotic during the photo inhibitory treatment.

Immunological quantification of the D 1 protein Polypeptides of the thylakoid membranes were separated by denaturing SDS-PAGE (Laemmli, 1970) with a 12 to 22.5% gradient of acrylamide and 4 M urea in the separating gel. After electrophoretic separation, the polypeptides were transferred to an Immobilon PVDF membrane (Millipore) and immunodetection of the D1 protein was performed with a chemiluminescence kit (Bio-Rad). The D1 protein was quantified by scanning the immunoblots with a laser densitometer (LKB).

RNA isolation, electrophoresis and Northern blot analysis Total RNA was isolated from Synechocystis 6803 cultures as described by Mohamed and Jansson (1989) with minor modifications. RNA was fractionated in agarose gels (1.2 % w/v) containing formaldehyde (2.2M). Transfer to a nylon membrane (Hybond N, Amersham), prehybridization, hybridization and washing conditions were chiefly according to the instruction manual (Amersham). The 0.8 kb HaeII-NcoI fragment of the psbA-2 gene of Syne· chocystis 6803 was used to probe the psbA messenger RNA. The probe was radioactively labeled by the random priming method (a kit from Amersham). Prehybridization and hybridization were done at 42°C in the presence of 50 % formamide (deionized with Amberlite). Membranes were exposed to X-ray films (Fuji New RX) with intensifying screens. A laser densitometer (LKB) was used to scan the auto radiographic signals. After removing the first probe (psbA) the same membrane was used in another hybridization (rrngene probe). The rrn genes of Anacystis nidulans from the pAN4 plasmid (Tomioka et al., 1981) were used as an internal standard to

Photoinhibition and D1 Protein Degradation in Synechocystis 6803 verify equal loading of the lanes and to confirm the size of the fulllength Synechocystis 6803 psbA mRNA (1.2 kb, Mohamed and Jansson, 1989).

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Oxygen evolution and Dl protein degradation in high light In the absence of antibiotics, 50 % inhibition of PS II activity in vivo was reached within 40 min at a PPFD of 1500~molm-2s-l, whereas in the presence of procaryotic protein synthesis inhibitors, the loss of PS II activity was clearly enhanced and 50 % inhibition was achieved within 20 min (Fig. 1). In pulse and chase experiments, the highest amount of radioactivity incorporated into the proteins of 32 kDa and 64kDa (Fig. 2 b). The 32kDa protein was identified as the Dl protein by using immunoblotting with D1 specific antiserum (data not shown). The D1 protein was the only one, that clearly disappeared during the high-light chase with a half-life of about 30 min. Presence of protein synthesis inhibitors only slightly affected the rate of D1 protein degradation (Fig. 2 a). In the presence of CAP, the half-life of the D1 protein was about 20 min, whereas in streptomycin- and rifampicin-treated cells the half-lives were about 40 and 50 min, respectively. In Synechocystis 6803, a half-life of 60 min has been reported for the D1 protein at a PPFD of 1000 ~mol m- 2 S-I by Ohad et al. (1992).

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Time (min) Fig. 1: Photo inhibition of PS II in low-light-grown Synechocystis 6803 cells in vivo. The cells were illuminated at a PPFD of 1500I-lmolm- 2 s- 1 in the presence or absence of protein synthesis inhibitors. Control (0), CAP (A), streptomycin (6), rifampicin (e). Photosynthetic oxygen evolution was measured using 0.25 mM DCBQ as an electron acceptor with 0.25 mM ferricyanide. The control value was 315 ± 91-lmol02 (mgChl)-lh- l. The data represent means ± SEM (n = 5).

Fig. 2: a) In vivo degradation of the D1 protein in Synechocystis 6803 cells at a PPFD of 1500 I-lmol m- 2 s- l. The cells were pulse-labeled with [3SS]L-methionine for 60 min and the chase period was carried out in the presence of non-radioactive L-methionine. Protein synthesis inhibitors were added at the beginning of the chase. Control (0), CAP (A), streptomycin (6), rifampicin (e). The values obtained for D1 protein were calculated as a percentage of pulse from densitometric scanning of autoradiograms using an area of stable polypeptides (37 - 45 kDa) as a reference. The data represent means of 2 independent experiments ± SEM. b) A typical autoradiogram of membrane proteins representing in vivo degradation of the D1 protein after 1 h eSS]L-methionine labeling of Synechocystis cells at a PPFD of 1500 I-lmol m -2 s -I. The samples were taken after O(A), 20(B), 40(C), 60(D) and 120(E) min of chase in high light in the presence of non-radioactive L-methionine.

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course of photoinhibition of PS II. Only a slight (about 15 %) net loss of the D1 protein occurred during strong illumination of Synechocystis cells (Fig. 3) in spite of serious photoinhibition of PS II. However, in the presence of procaryotic protein synthesis inhibitors, a rapid loss of the D1 protein from the thylakoid membranes was observed in the course of illumination. In the presence of CAP, the rate of D1 protein degradation was slightly faster than in the presence of streptomycin or rifampicin, as noticed also in pulse and chase experiments (Fig. 2 a).

Effect ofstrong light and protein synthesis inhibitors on the level ofpsbA mRNA The high light treatment caused a significant accumulation of the psbA mRNA in low-light-grown cells (Table 1, Fig. 4). Almost a three-fold increase in the amount of the psbA mRNA was observed within 45 min, when compared with the level at the beginning of the experiment (time 0). Addition of CAP to the cell suspension at the beginning of the high light treatment efficiently inhibited the accumulation of the psbA mRNA and a zone of degradation products Table 1: Effects of protein synthesis inhibitors on the light-stimulated accumulation of psbA transcripts. Low-light-grown Synecho· cystis 6803 cells were transferred to a PPFD of 1500 J.lmol m- 2s- 1 for 45 min. Total RNA was extracted, fractionated, blotted and hybridized to a 32P-Iabeled psbA gene probe. Autoradiograms were scanned by a laser densitometer. Relative values are calculated using 165 rRNA as an internal standard, mean ± 5EM, n = 2. One of the autoradiograms is presented in Fig. 4.

Fig.3: Loss of the Dl protein from photosynthetic membranes of Synechocystis 6803 during illumination at a PPFD of 1500).1molm- 2 s- 1 in the presence and absence of protein synthesis inhibitors. The amount of the D1 protein was determined by quantitative immunoblotting. a) Immunological quantification of the Dl protein. Immunoblot and a graphic representation of a densitometrically quantified blot indicating linearity of the response. Different amounts of control membranes (indicated as ).1g ChI in the blot) were used in different wells. b) Effect of photoinhibitory illumination on the amount of the D1 protein in the photosynthetic membranes in the presence and absence of antibiotics. Lanes 1, 2 and 3 represent control cells illuminated for 0, 45 and 90 min, respectively; lanes 4 and 5 = cells illuminated in the presence of CAP for 45 and 90 min; lanes 6 and 7 = in the presence of streptomycin for 45 and 90 min; lanes 8 and 9 = in the presence of rifampicin for 15 and 60 min; lanes 10, 11 and 12 = as lanes 1, 2 and 3, but from a different, independent experiment. c) Graphic representation of the loss of the Dl protein from photosynthetic membranes of Synecho. cystis 6803 during photo inhibitory illumination. Control (0), CAP (A.), streptomycin (6), rifampicin (.). In the rifampicin experiments, the cells were pretreated with rifampicin (500 J.lg mL -I) for 30 min in photoinhibitory conditions to deplete the psbA mRNA pool (half-life of the psbA mRNA = 15 min). 3.5 J.lg chlorophyll was applied to each well. The data represent means of 2 - 3 independent experiments ± 5EM.

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Photoinhibition and Dl Protein Degradation in Synechocystis 6803

(0.7 -0.9 kb) of the psbA mRNA was formed (Fig. 4, lane 3). In the presence of streptomycin, the psbA mRNA accumulation was also clearly inhibited as compared with the control cells; however, the level of psbA transcripts was higher than in the presence of CAP (Fig. 4, lane 4). The accumulation of the psbA mRNA was completely blocked by rifampicin, an inhibitor of transcription (Fig. 4, lane 5). Chase of the amount of psbA transcripts after addition of rifampicin gave a half-life of 15min at a PPFD of 1500J.lmolm- 2 s- 1 (data not shown). Discussion

It is well documented that photo inhibition of PS II is accelerated in the presence of procaryotic protein synthesis inhibitors (Ohad et aI., 1984; Samuelsson et aI., 1985; Schuster et aI., 1988; Wi.inschmann and Brand, 1992). This has been attributed to a block in the recycling of the D1 protein that is damaged and degraded during photoinhibition. However, there are only few in vivo studies concentrating on the actual relationship between the D1 protein degradation and photoinhibition of PS II in the presence and absence of procaryotic protein synthesis inhibitors (Schuster et aI., 1988). Synechocystis cells exposed to strong light (1500 J.lmol m- 2 s- 1) without protein synthesis inhibitors seem to reach a steady-state level of suppressed (about 75 %) PS II activity during illumination (Fig. 1). This result suggests an equilibrium state between photo inhibition and a process repairing photodamaged PS II centers (Wi.inschmann and Brand, 1992). However, only a portion of PS II activity (about 25 %) could be maintained in Synechocystis cells at a PPFD of 1500 J.lmol m- 2 s- 1• When protein synthesis was inhibited, PS II activity was totally lost within two hours indicating that de novo protein synthesis is indeed necessary for maintaining this low PS II activity via de novo synthesis of the D1 protein. When de novo protein synthesis and thus the repair of PS II centers are inhibited, degradation of the D1 protein proceeds rapidly and closely follows the rate of photoinhibition of PS II electron transport in Synechocystis cells illuminated at a PPFD of 1500J.lmolm- 2 s- 1• Fast D1 protein degradation is demonstrated in the presence of protein synthesis inhibitors both by the chase of radioactivity in [35 S]met-labeled D1 protein under photoinhibitory illumination (Fig. 2 a) and by immunoblotting of the D1 protein from membranes isolated from samples taken in the course of the photoinhibitory treatment (Fig. 3). These results are in agreement with the finding made earlier by our group concerning higher plants: if there is plenty of non-appressed thylakoid area for repairing photodamaged D1 protein, its degradation occurs more readily than in tightly stacked chloroplasts (Kettunen et aI., 1991; Tyystjarvi et aI., 1992). In spite of severe photoinhibition of PS II and fast degradation of the D1 protein, only a slight net loss (15 %) of the D1 protein could be observed during the high light treatment (Figs. 1-3). In Chlamydomonas reinhardtii cells, a reduction of 20 % was observed in the total amount of the D1 protein after 90 min of photo inhibitory treatment, while 80 % of PS II activity was lost (Schuster et aI., 1988). Recent studies with

673

higher plants have also shown that no significant net loss of the D1 protein occurs under photo inhibitory conditions (Cleland et aI., 1990; Kettunen et aI., 1991; Schnettger et aI., 1993) provided that high light stress is not prolonged (Kettunen et aI., 1991; Adamska et aI., 1992). Our results indicate that synthesis of the D1 protein in Synechocystis cells can almost match the rate of its degradation even under high light stress, and it seems likely that reassembly and activation of PS II after insertion of a new copy of the D1 protein to the PS II complex are the rate-limiting factors for the repair of PS II. The relatively slow process of reassembly and/or activation of PS II may thus explain why only 25 % of PS II activity can be detected in high light without a significant net loss of the D1 protein. Fast degradation of the D1 protein in cells with no appressed thylakoids and the efficiency of both degradation and synthesis of the D1 protein made us hypothesize that probably the contact of psbA mRNA-carrying polyribosomes with thylakoid membranes is involved in regulation of degradation of the D1 protein. Using different procaryotic protein synthesis inhibitors, we could induce conditions in which thylakoids were fully loaded with polyribosomes (using CAP) (Chua et aI., 1976; Vasquez, 1979; Herrin and Michaels, 1985) or thylakoids were depleted of polyribosomes (using rifampicin, an inhibitor of procaryotic transcription). According to our results polyribosomes do not seem to have a significant role in regulation of the rate of D1 protein degradation (Figs. 2 and 3). Since only a minor net loss of the D1 protein from thylakoid membranes could be detected, degradation and synthesis of the D1 protein must occur more or less at the same rate. However, these two processes do not seem to be directly synchronized, since degradation proceeds readily while synthesis is inhibited. Thus, protein synthesis activity does not seem to be among the factors that directly regulate the rate of D1 protein degradation. CAP has been widely used in photoinhibition research, especially to test whether de novo synthesis is involved in the recovery of photosynthetic activity after a photoinhibitory treatment (Ohad et aI., 1984; Schuster et aI., 1988; Kirilovsky et aI., 1990). Use of CAP in such experiments has been criticized (Okada, 1991). According to our results, the effect of another common translation inhibitor, streptomycin, on the rate of inhibition of PS II electron transport (Fig. 1), on the rate of D1 degradation and on the steady-state level of the D1 protein (Figs. 2 and 3) is almost similar to that of CAP, when Synechocystis 6803 cells are exposed to strong illumination. However, slightly faster degradation of the D1 protein was noticed in the presence of CAP as compared with streptomycin. Moreover, degradation products of the psbA mRNA also existed in the presence of CAP (Fig. 4, lane 3). These findings could be explained by effects of oxygen or other free radicals, the generation of which could possibly be accelerated during strong illumination in the presence of CAP (Okada, 1991). An interesting observation is that either transcription of the psbA genes or stability of the psbA mRNA is regulated by de novo synthesized protein factors. This conclusion is based on the experiments showing that the amount of the psbA mRNA increased almost three-fold upon transfer of Synecho· cystis cells to strong light, whereas only a minor accumula-

674

EIRA KANERVO, PIRKKO MAENPAA, and EVA-MARr ARO

tion of these transcripts occurred in the presence of procaryotic translation inhibitors (Table 1, Fig. 4). A high-lightinduced psbA mRNA accumulation in cyanobacterial cells was also demonstrated earlier (Mohamed and Jansson, 1989; Schaefer and Golden, 1989; Kulkarni et al., 1992). There are two possible explanations for the light-stimulated psbA mRNA accumulation: either stimulation of transcription or increased stability of the transcripts. In both cases regulative proteins may be involved. In chloroplasts, mRNA binding proteins of nuclear origin have been reported to be involved in stabilizing the psbA mRNA or acting as translational activators (Danon and Mayfield, 1991; Schuster and Gruissem, 1991). Also in cyanobacteria, accumulation of psbAII and psbAIII mRNA in Synechococcus 7942 cells in high light is probably a transcriptional response (Kulkarni et al., 1992). Moreover, synthesis of a degradation factor decreasing the stability of the psbAI transcripts has also been reported in Synechococcus 7942 cells subjected to strong illumination (Kulkarni et al., 1992). Our finding of high-light-induced accumulation of the psbA mRNA and suppression of this accumulation in the presence of translation inhibitors suggests the existence of protein factor(s) induced under high light conditions and involved in the regulation of the expression of the psbA genes in Synechocystis 6803. Whether it is a question of an alteration in transcription activity or in mRNA stability remains to be elucidated. Acknowledgements

We want to thank Prof. I. Ohad for the Dl antibody and Virpi Paakkarinen for skillful help in Western blotting. This work was supported by the Academy of Finland, Ministry of Agriculture and Forestry and the Jenny and Antti Wihuri Foundation.

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